The present invention relates to methods for treating movement disorders. In particular, the present invention relates to methods for treating epilepsy by intracranial administration of a botulinum toxin.
Intracranial Drug Delivery
A major impediment to therapeutic treatment of a neurodegenerative disease, such as various movement disorders, is the blood-brain barrier which significantly limits penetration of the brain by even small molecules from the bloodstream upon peripheral administration of a pharmaceutical. To circumvent the blood-brain barrier direct infusion of various bioactive substances has been carried out. Most clinical experience is with intraventricular (i.e. into a cerebral-spinal fluid [CSF] filled ventricle of the brain) drug delivery. Thus, ventricular infections have been treated by direct infusion of antibiotic. Additionally, intraventricular infusion: of baclofen to treat spasticity; various chemotherapeutics, radiolabelled antibodies, and cytokines to treat brain tumors; cholinergic agonists and Nerve Growth Factor (NGF) to treat Alzheimer's disease, and; dopamine to treat Parkinson's disease is known. Unfortunately, there is a brain-CSF barrier such that penetration of drugs into brain tissue from CSF is suboptimal. Intraventricular drug delivery has therefore been met with limited success in the treatment of, for example, solid tumors, neurodegenerative diseases (such as movement disorders) and other intraparenchymal pathology.
The drawbacks and deficiencies of intraventricular drug delivery has led to increasing interest in direct infusion of drugs into brain parenchyma. Administration of therapeutic bioactive substances to various brain sites of interest has be achieved with reproducible submillimeter precision using modern stereotactic techniques. Thus, intraparenchymal infusion of lidocaine, muscimol and NGF have been used to treat Parkinson's disease, and intraparenchymal infusion of KCL has been used to treat epilepsy. The most widespread use of direct intracerebral clinical administration of a bioactive substance has been to treat brain tumors, which has included gene therapy by delivery of one or more therapeutic genes into brain tumor cells. See e.g. Kaplitt M. G. et al., Surgical drug delivery for neurodegenerative diseases. Clin Neurosurg 48; 127-144: 2001. The localized microinjection of lidocaine and muscimol has been found to selectively inactivate focal neuronal activity in the subthalamic nucleus of Parkinson's disease patients (Levy R. et al., Lidocaine and muscimol microinjections in subthalamic nucleus reverse Parkinsonian symptoms, Brain 2001 October; 124(Pt 10):2105-18). The effect is transient and is directly attributable to intrinsic activity and pharmacological half-lives of these agents.
Additionally, patients with malignant gliomas have been treated with a chimeric toxin composed of the cytokine, IL-4, and Pseudomonas exotoxin, administered through stereotactic catheter implantation into tumors through small-twist drill holes. Despite varying volumes of infusion, no profound neural or systemic toxicity resulted. Rand R. W. et al., Intratumoral administration of recombinant circularly permuted interleukin-4-Pseudomonas exotoxin in patients with high-grade glioma, Clin Cancer Res 6; 2157-2165: 2000.
Thus, precise intracranial therapeutic delivery of bioactive macromolecules can be achieved through stereotactic methodologies. When a molecule is introduced into the extracellular space in brain tissue, because of the narrowness and irregularity of channels, it diffuses through the tissue at a rate which is at least twelve times slower than diffusion of the molecule through agar. Penn R., The future of CNS infusion systems, chapter 218, pages 2073-2076 of Gildenberg, P., Textbook of Stereotactic and Functional Neurosurgery, McGraw-Hill (1998). Due to take up by cells, binding of receptors or extracellular matrix, enzymatic degradation, or elimination by the vascular system it is unusual for a bioactive molecule applied as a point source to diffuse in brain tissue more than a few millimeters from its' site of administration. Ibid. For functional stereotactic surgeons the type of applications most suited for intraparenchymal drug application are those in which a small lesion or local electrical stimulation has already proved effective, such as for the treatment of tremor. Thus, it has been postulated that the use of selective neurotransmitter agents or antagonists may be more effective to inhibit specific neurons than is current therapies of deliberately making or inducing a lesion (i.e. by radiation, thermal, cryo or electrical ablation or surgical incision) that indiscriminately destroys neurons and axons. Ibid.
Movement Disorders
A movement disorder is a neurological disturbance that involves one or more muscles or muscle groups. Movement disorders include Parkinson's disease, Huntington's Chorea, progressive supranuclear palsy, Wilson's disease, Tourette's syndrome, epilepsy, and various chronic tremors, including essential tremor, tics and dystonias. Different clinically observed movement disorders can be traced to the same or similar areas of the brain. For example, abnormalities of basal ganglia (a large cluster of cells deep in the hemispheres of the brain) are postulated as a causative factor in diverse movement disorders.
Tremors are characterized by abnormal, involuntary movements. An essential tremor is maximal when the body part afflicted (often an arm or hand) is being used, for example when attempts at writing or fine coordinated hand movements are made. Typical chemotherapy is use of the drug propranolol (Inderal) which has the side effects of low blood pressure and heart rate changes. A resting tremor is common in Parkinson's disease and in syndromes with Parkinsonian features. A resting tremor is maximal when the extremities are at rest. Often, when a patient attempts fine movement, such as reaching for a cup, the tremor subsides. Systemic anticholinergic medications have been used with some success.
Dystonias are involuntary movement disorders characterized by continued muscular contractions which can result in twisted contorted postures involving the body or limbs. Causes of dystonia include biochemical abnormalities, degenerative disorders, psychiatric dysfunction, toxins, drugs and central trauma. Thalamotomy and/or subthalamotomy or campotomy are currently the preferred neurosurgical procedures to treat dystonia, and are carried out with techniques and brain targets similar to the surgical treatment of Parkinson's disease. Tasker R., Surgical Treatment of the Dystonias, chapter 105, pages 1015-1032, in Gildenberg P. L. et al., Textbook of Stereotactic and Functional Neurosurgery, McGraw-Hill (1998).
Particular dystonias can include spasmodic torticolis, blepharospasm and writer's cramp. Spasmodic torticollis is a syndrome that usually affects adults, and involves the involuntary turning of the neck to one side. Some individuals may not even notice initially that the head and neck are turned. Blepharospasm is an involuntary movement which involves intermittent forceful closure of the eyelids. Writer's cramp is a cramping abnormal posture which develops when one is writing, or performing other actions with the hands. Symptoms may progress to involve the arm and shoulder.
Tic disorders (including Tourette's) are usually very rapid, short lived stereotyped repeated movements. The more common tics involve the motor systems, or are vocal in nature. Motor tics often involve the eyelids, eyebrows or other facial muscles, as well as the upper limbs. Vocal tics may involve grunting, throat clearing, coughing or cursing. Individuals with tic disorders will often describe a strong urge to perform the particular tic, and may actually feel a strong sense of pressure building up inside of them if the action is not performed. For example, a motor tic that may involve the abrupt movement of one of the arms may be controllable for a short period of time if the affected person sits on his hands; however, the almost irresistible urge to do the action often takes over and result in the tic action.
Tourette's syndrome is a tic disorder which often begins in childhood or adolescence and is much more common in males. There are both multiple motor tics, as well as vocal tics present. The tics often change from involvement of one body part to another, and the disease often gets better and worse intermittently, with periods of almost minimal activity, and other times when some patients have difficulty functioning. Other neurobehavioral difficulties often accompany the syndrome. These include attention deficit hyperactivity disorder (ADHD) and obsessive-compulsive disorder. Treatment of most tic disorders employs the use of medications that decrease the amount of dopamine in the brain, such as dopamine antagonists. Unfortunately these drugs are associated with side effects such as other movement disorders, including Parkinsonism (stiffness, slow movement and tremors). In addition to Tourette's syndrome, tics may be associated with head injury, carbon monoxide poisoning, stroke, drug use and mental retardation.
Progressive supranuclear palsy is a movement disorder in which patients have significant difficulty moving their eyes vertically (up and down) initially, followed by all eye movements become limited (ophthalmoplegia). Patients can also develop dementia, rigidity, bradykinesia (slow movements) and a propensity for falls.
Huntington's chorea is a genetically inherited disorder that has both neurological and psychiatric features. Most cases develop when people are in their forties or fifties, but early and late onset is also possible. The disease may begin with either the neurological or mental status changes. The neurological symptoms may vary, but include chorea. Chorea (derived from a Greek word meaning, “to dance”) is a series of movements that is dance-like, jerky, brief, and moves from one part of the body to another. Clumsiness, fidgetiness and jumpiness may also occur. Facial movements, especially around the jaw, may occur. There is often difficulty with walking and posture. The psychiatric symptoms may present as paranoia, confusion, or personality changes. As the disease progresses, a significant dementia develops. MRI brain imaging may show atrophy (shrinkage) of a portion of the basal ganglia (involved in movement) that is known as the caudate nucleus.
Wilson's disease is a disorder that involves the nervous system and liver function. The neurological problems include tremors, incoordination, falling, slurred speech, stiffness and seizures. Psychiatric problems can occur and patients can develop severe liver damage if this affliction is untreated. Elevated copper and ceruloplasmin levels are diagnostic.
Unfortunately, a movement disorder, including those set forth above, can become resistant to drug therapy. Drug resistant tremors can include resting tremors, such as can occur in Parkinson's disease, and action tremors, such as essential tremor, multiple sclerosis tremors, post traumatic tremors, post hemiplegic tremors (post stroke spasticity), tremors associated with neuropathy, writing tremors and epilepsy.
Parkinson's Disease
Parkinson's disease is a movement disorder of increasing occurrence in aging populations. Parkinson's disease is a common disabling disease of old age affecting about one percent of the population over the age of 60 in the United States. The incidence of Parkinson's disease increases with age and the cumulative lifetime risk of an individual developing the disease is about 1 in 40. Symptoms include pronounced tremor of the extremities, bradykinesia, rigidity and postural change. A perceived pathophysiological cause of Parkinson's disease is progressive destruction of dopamine producing cells in the basal ganglia which comprise the pars compartum of the substantia nigra, a basal nuclei located in the brain stem. Loss of dopamineric neurons results in a relative excess of acetylcholine. Jellinger, K. A., Post Mortem Studies in Parkinson's Disease—Is It Possible to Detect Brain Areas For Specific Symptoms?, J Neural Transm 56 (Supp); 1-29:1999.
Parkinson's disease is a progressive disorder which can begin with mild limb stiffness and infrequent tremors and progress over a period of ten or more years to frequent tremors and memory impairment, to uncontrollable tremors and dementia.
Drugs used to treat Parkinson's disease include L-dopa, selegiline, apomorphine and anticholinergics. L-dopa (levo-dihydroxy-phenylalanine) (sinemet) is a dopamine precursor which can cross the blood-brain barrier and be converted to dopamine in the brain. Unfortunately, L-dopa has a short half life in the body and it is typical after long use (i.e. after about 4-5 years) for the effect of L-dopa to become sporadic and unpredictable, resulting in fluctuations in motor function, dyskinesias and psychiatric side effects. Additionally, L-dopa can cause B vitamin deficiencies to arise.
Selegiline (Deprenyl, Eldepryl) has been used as an alternative to L-dopa, and acts by reducing the breakdown of dopamine in the brain. Unfortunately, Selegiline becomes ineffective after about nine months of use. Apomorphine, a dopamine receptor agonist, has been used to treat Parkinson's disease, although is causes severe vomiting when used on its own, as well as skin reactions, infection, drowsiness and some psychiatric side effects.
Systemically administered anticholinergic drugs (such as benzhexol and orphenedrine) have also been used to treat Parkinson's disease and act by reducing the amount of acetylcholine produced in the brain and thereby redress the dopamine/acetylcholine imbalance present in Parkinson's disease. Unfortunately, about 70% of patients taking systemically administered anticholinergics develop serious neuropsychiatric side effects, including hallucinations, as well as dyskinetic movements, and other effects resulting from wide anticholinergic distribution, including vision effects, difficulty swallowing, dry mouth and urine retention. See e.g. Playfer, J. R., Parkinson's Disease, Postgrad Med J, 73; 257-264:1997 and Nadeau, S. E., Parkinson's Disease, J Am Ger Soc, 45; 233-240:1997.
Before the introduction of L-dopa in 1969, stereotactic surgery offered one of the few effective treatments for Parkinson's disease. The significant known deficiencies and drawbacks associated with therapeutic drugs to treat Parkinson's disease, including the long term limitations of L-dopa therapy have led to renewed interest in neurosurgical intervention. Unilateral stereotactic thalamotomy has proven to be effective for controlling contralateral tremor and rigidity, but carries a risk of hemiparesis. Bilateral thalamotomy carries an increased risk of speech and swallowing disorders resulting. Stereotactic pallidotomy, surgical ablation of part of the globus pallidus (a basal ganglia), has also be used with some success. Aside from surgical resection, high frequency stimulating electrodes placed in the ventral intermedialis nucleus has been found to suppress abnormal movements in some cases. A variety of techniques exist to permit precise location of a probe, including computed tomography and magnetic resonance imaging. Unfortunately, the akinesia, speech and gait disorder symptoms of Parkinson's disease are little helped by these surgical procedures, all of which result in destructive brain lesions.
Epilepsy
Epilepsy is the most common serious neurological disorder (Shorvon, S., Epidemiology, classification, natural history, and genetics of epilepsy, Lancet 1990 Jul. 14; 336(8707):93-6; McNamara J., The neurobiological basis of epilepsy, Trends Neurosci 1992 October; 15(10):357-9. A seizure is a neurological dysfunction which results from abnormal, excessive, hypersynchronous discharges from an aggregate of central nervous system neurons. A seizure can be manifested behaviorally (if motor systems are involved) or electrographically. Epilepsy describes a condition in which a person has recurrent seizures due to a chronic, underlying process. Although there are various epilepsy syndromes in which the clinical and pathologic characteristics differ the common underlying etiology is neuronal hyperexcitability. Thus, epilepsy encompasses disorders of central nervous system (CNS) hyperexcitability, characterized by chronic, recurrent, paroxysmal changes in neurological function that can be categorized according to electroencephalographic and clinical presentation (Dichter M., Basic mechanisms of epilepsy: targets for therapeutic intervention, Epilepsia 1997; 38 Suppl 9:S2-6).
Excluding neonatal febrile seizures, the estimated occurrence of epilepsy in the general population is about 0.5%-1% (Barnes D., Debate about epilepsy: what initiates seizures?, Science 1986 Nov. 21; 234(4779):938-40, erratum in Science 1987 Jan. 2; 235(4784):16; Rogawski M., et al., Antiepileptic drugs: pharmacological mechanisms and clinical efficacy with consideration of promising developmental stage compounds, Pharmacol Rev 1990 September; 42(3):223-86). Severe, penetrating head trauma is associated with up to a 50% risk of leading to epilepsy. Other causes of epilepsy include stroke, infection and genetic susceptibility.
While recurrent seizures are a hallmark of epilepsy, isolated, nonrecurrent seizures can occur in otherwise healthy individuals for a variety of reasons, such as poisoning, and such individuals are not considered to have epilepsy (Dichter 1997, Ibid). Epileptic seizures are broadly categorized into two groups: focal (partial) and generalized seizures. Focal seizures arise from abnormal activity of a limited group of neurons in cortical or subcortical regions of the brain. The underlying structural abnormality or lesion can develop as a result of birth injury, head trauma, tumor, abscess, infarction, vascular malformation or genetic disease (Dichter 1997, Ibid). The location of the focal activity can be identified by the clinical seizure presentation or may be cryptic. Equivalently, the active focus may not involve the lesion itself but may arise in adjacent or distant (but connected) neuronal populations, supporting the hypothesis of plastic synaptic reorganization underlying focal hyperexcitability. See e.g. Prince D. A., Epileptogenic neurons and circuits. In: Jasper's Basic Mechanisms of the Epilepsies, Third Edition (1999), Delgado-Escueta A. V., et al., editors), Advances in Neurology 79: 665-684.
Focal seizures are termed “simple” if there is no apparent change in consciousness, otherwise they are termed “complex”. Complex focal seizures involve the temporal lobe and limbic system, and are the most common manifestation of epilepsy in adults. Focal seizures that spread to become bilateral electrographically, with concomitant loss of consciousness and with or without motor manifestations, are said to be secondarily generalized. Primary generalized seizures initiate with bilateral electrographic activity, loss of consciousness, and with or without motor convulsions. Focal epilepsy can involve almost any part of the brain and usually results from a localized lesion of functional abnormality. One type of focal epilepsy is the psychomotor seizure. Current therapy for focal epilepsy includes use of an EEG to localize abnormal spiking waves originating in areas of organic brain disease that predispose to focal epileptic attacks, followed by surgical excision of the focus to prevent future attacks.
While the etiology of epilepsy can be multiple, the pathophysiology of epilepsy (in terms of the generation of synchronized neuronal activity) is thought to consistently reflect both fundamental changes in basic neuronal physiological properties together with enhanced synaptic plasticity. The process of epileptogenesis therefore involves a limited range of intrinsic cellular changes that lead to neuronal imbalances in net excitation or inhibition, or in the enhanced excitatory coupling of neuronal aggregates (Prince 1999 ibid, and; Prince D. A., Cellular mechanisms of interictal-ictal transitions. In: Mechanisms of Epileptogenesis. The Transition to Seizure, Dichter M. A., editor, Plenum Press, New York, 57-71 (1998)). The hallmark of epileptogenesis is the appearance of interictal (between seizure) bursts or discharges. This pattern of activity is observed on the EEG record as brief (80-200 millisecond), large, sharp spikes against an otherwise normal background of activity (McCormick and Contreras 2001, supra). These periodic EEG spikes correlate to prolonged cellular depolarizations (paroxysmal depolarization shift, PDS), while the quiescent periods reflect phases of cellular hyperpolarization.
Thus it is believed that distinct neural circuitry underlying the initiation and propagation of seizures can be identified for each type of epileptic seizures because seizures are manifestations of abnormal activity in neuronal networks that normally are engaged in routine physiological processes (Gale K., Focal trigger zones and pathways of propagation in seizure generation. In: Epilepsy: Models, Mechanism and Concepts, Schwartzkroin P. A., editor, Cambridge University Press, U.K., pages 48-93 (1993)).
While there may be no dedicated “seizure circuit” within the brain, seizure propagation clearly results from, and depends upon, the specific neuroanatomy of the interconnected neuronal circuitry. It is well established that some parts of the brain, such as the limbic system, are more susceptible to epileptogenesis and seizure propagation than are other areas, such as the neocortex. The hippocampus (a part of the limbic system) has been extensively studied, as much for its orderly and accessible cellular architecture as for its tendency to be become epileptogenic when provided an appropriate stimulus, both in vitro and in vivo. The epileptogenic hippocampus displays significant synaptic reorganization and changes in plasticity that potentiate hyperexcitability.
Mossy fiber sprouting, secondary to loss of target cells in Ammon's horn area CA3 of the hippocampus, and subsequent establishment of synapses in the inner molecular layer of the normally hypoexcitable dentate gyrus (fascia dentata), results in recurrent (feedback) excitation (Sutula T. P., Sprouting as an underlying cause of hyperexcitability in experimental models and in the human epileptic temporal lobe. In: Epilepsy: Models, Mechanism and Concepts, Schwartzkroin P. A. editor, Cambridge University Press, U.K., pages 304-322 (1993)). This synchronized hippocampal activity directly results from phasic imbalances between excitatory and inhibitory neuronal populations. The hippocampus, together with the amygdala, have been implicated in human temporal lobe epilepsy, an often intractable condition and the most common epileptic disorder in adults. Cellular degeneration in the hippocampus (termed hippocampal sclerosis), and compensatory changes in wiring, are a prime cause of hyperexcitability and a rationale for resective surgery in the treatment of temporal lobe epilepsy. As previously noted, the neocortex is less vulnerable to seizure propagation from local foci as compared to the limbic system. This self-limiting capacity may arise from the inherent characteristic of selective presynaptic depression of excitatory transmission under conditions of high frequency firing (Galaretta M. et al., Frequency-dependent synaptic depression and the balance of excitation and inhibition in the neocortex, Nature Neurosci 1(7); 587-594: 1998), as would occur during seizure propagation. Changes in the connectivity and communication between neuronal cells (neural plasticity) have been implicated in the pathophysiology of epilepsy. Synaptic plasticity refers to characteristic activity-dependent changes in synaptic efficacy that may either produce enhancement (long term potentiation, LTP) or inhibition (long term depression). The observed changes may reflect altered functionality at presynaptic and postsynaptic locations. Presynaptic changes, which involve alterations in the kinetics of synaptic vesicle recycling, affect the rate of neurotransmission and, therefore, of synaptic activity (Staley K., et al., Presynaptic modulation of CA3 network activity, Nat Neurosci 1998 July; 1(3):201-9, erratum in Nat Neurosci 1998 August; 1(4):331; Wang L., et al., High-frequency firing helps replenish the readily releasable pool of synaptic vesicles, Nature 1998 Jul. 23; 394(6691):384-8; Zakharenko S., et al., Visualization of changes in presynaptic function during long-term synaptic plasticity, Nat Neurosci 2001 July; 4(7):711-7). Synchronization of presynaptic and postsynaptic activity can also enhance the efficacy of synaptic transmission and facilitate LTP (Ganguly K., et al., Enhancement of presynaptic neuronal excitability by correlated presynaptic and postsynaptic spiking, Nat Neurosci 2000 October; 3(10):1018-26). Postsynaptic changes in synaptic plasticity have been suggested as the basis for such processes as learning and memory and may contribute, in part, to the process of epileptogenesis.
This neuroanatomical basis for brain neuronal network connectivity, and the mechanisms of epileptogenesis have been investigated, for example, in the kindling model of focal, temporal lobe (limbic) epilepsy. In the kindling paradigm, test animals are subjected to repeated, focal electrical stimulation through bipolar, stereotactically-implanted depth electrodes (Goddard G. V. et al, A permanent change in brain function resulting from daily electrical stimulation. Exp Neurol 25(3); 295-330:1969). The electrodes are typically placed in either the amygdala or dorsal hippocampus of rats. The initially subconvulsive stimulations gradually precipitate a described progression of behavioral manifestations, which are then scored (Racine R. J., Modification of seizure activity by electrical stimulation. II. Motor seizure, Electroencephalogr Clin Neurophysiol. 32(3); 281+-94:1972b), and which correlate to the gradual electrographic appearance of afterdischarges (episodic, ictal, electrical activity in the absence of exogenous stimulus). Together with their appearance, the threshold electrical stimulus required to produce an afterdischarge gradually lowers, paralleling the seizure threshold drop seen in human epileptogenesis. The kindling process fails to develop in the absence of afterdischarges (Racine R. J., Modification of seizure activity by electrical stimulation. I. After-discharge threshold, Electroencephalogr Clin Neurophysiol. 32(3); 269-79:1972a). Eventually, the continued repeated stimulations precipitate full motor seizures (Goddard et al, 1969, supra). Once established, the lowered seizure threshold and hyperexcitability are permanent, such that an otherwise subconvulsive (threshold) stimulus at a later time point triggers a generalized, convulsive seizure. It should be noted that the generalized seizure is simply an end point, reflecting recruitment of motor output pathways, and does not in and of itself establish the kindled phenotype. Rather, kindling reflects the whole process of population recruitment and spread by repeated focal afterdischarge.
Antiepileptic drug (“AED”) therapy is the mainstay of treatment for most patients with epilepsy and a variety of drugs have been used. See e.g., Fauci, A. S. et al., Harrison's Principles of Internal Medicine, McGraw-Hill, 14th Edition (1998), page 2321. About twenty percent of patients with epilepsy are resistant to drug therapy despite efforts to find an effective combination of antiepileptic drugs. Surgery can then be an option. Thus, patients with refractory epilepsy and intractable seizures can be candidates for surgical resection followed by up AED therapy. Surgery is superior to long-term drug treatment alone in the management of temporal lobe epilepsy (Wiebe et al, 2001, supra). Typically, video-electroencephalogram (EEG) monitoring is used to broadly define the anatomic location of the seizure focus and to correlate the abnormal electrophysiological activity with behavioral manifestations of the seizure. Scalp or scalp-sphenoidal recordings are usually sufficient for localization. Functional imaging studies such as SPECT and PET, as well as direct electrophysiological analysis (subdural or depth electrode mapping) are adjunctive tests that can help verify the localization of an apparent epileptogenic region with an anatomic abnormality. A high resolution MRI scan is routinely used to identify structural lesions. Once the presumed location of the seizure onset is identified, additional studies, including neuropsychological testing and the intracarotid amobarbital test (Wada's test) can be used to assess language and memory localization and to determine the possible functional consequences of surgical removal of the epileptogenic region. In some cases, the exact extent of the resection to be undertaken can be determined by performing cortical mapping at the time of the surgical procedure. This involves electrophysiologic recordings and cortical stimulation of the awake patient to identify the extent of epileptiform disturbances and the function of the cortical regions in questions. Clearly, consideration for surgery is contingent upon the identified and implicated substructure being resectable. The most common surgical procedures for patients with temporal lobe epilepsy are resection of the anteromedial temporal lobe (temporal lobectomy, which includes the nterolateralneocortex and the deeply located older structures—corticoamygdalohippocampectomy), or a more limited removal of the underlying hippocampus and amygdala (selective amygdalohippocampectomy. A third procedure, the selective neocortical resection, is rarely used, although focal seizures arising from extratemporal regions may be suppressed by a focal neocortical resection.
While invasive, surgery has proven to be superior to long-term drug treatment alone in the management of temporal lobe epilepsy (Wiebe S. et al, A randomized, controlled trial of surgery for temporal-lobe epilepsy, N Engl J Med 345(5): 311-8:2001). Unfortunately, about 5% of patients can still develop clinically significant complications from surgery and up to 10% of temporal lobectomy patients can still have seizures. Kwan P. et al, Refractory epilepsy: a progressive, intractable but preventable condition? Seizure 11: 77-84:2002). In cases where subcortical structures encompass, or are in proximity to, critical functional areas, and thus cannot be approached with standard resective techniques, the treatment regimen can be a maintenance of multiple AEDs for suppression of seizure activity (Kwan et al 2002, supra).
Stereotactic surgical procedures have been refined and aided by improvements in current imaging techniques, permitting precise identification and targeting of intracranial substructures, compared to more traditional ventriculography. MRI methods allow for clear and accurate definition, three-dimensional spatial orientation and structure localization. See e.g. Landi A. et al., Accuracy of stereotactic localization with magnetic resonance compared to CT scan: experimental findings, Acta Neurochir (Wien) 143; 593-601: 2001. As compared to historically traditional craniotomy and resective surgery, stereotactic surgery (as with, for example, refractive temporal lobe epilepsy) may be better tolerated by patients and more economical to perform. Parrent A. G. et al., Stereotactic surgery for temporal lobe epilepsy, Can J Neurol Sci 27(Suppl 1); S79-S84: 2000. In addition to guiding surgical resections, stereotactic techniques can be employed, alternatively, to specifically target a selected structure and deliver an effective therapy. While all surgical procedures are invasive and irreversible, stereotactically targeted therapies may be either invasive or noninvasive, and either irreversible or reversible.
Stereotactic radiofrequency ablation (invasive, irreversible) has been described for the treatment of intractable gelastic seizures arising from a hypothalamic hamartoma following MR imaging, focus localization and reference-based guidance of the lesioning electrode to the hypothalamus. See e.g. Parrent A. G., Stereotactic radiofrequency ablation for the treatment of gelastic seizures associated with hypothalamic hamartoma: case report, J Neurosurg 91: 881-884: 1999. Similarly, and in a manner analogous to that applied to the treatment of Parkinson's disease, stereotactic stimulation at high radiofrequency (130 Hz) of the subthalamic nucleus (STN, corpus luysi) has shown efficacy in seizure control in the absence of apparent anatomical lesioning. In the latter case, the STN was not the focus per se and the stimulation protocol affected the network propagation of seizures rather than the source focus itself. Benabid A. L. et al., Deep brain stimulation of the corpus luysi (subthalamic nucleus) and other targets in Parkinson's disease. Extension to new indications such as dystonia and epilepsy, J Neurol 248(Suppl 3); 37-47: 2001.
Stereotactic radiosurgery (radiotherapy) is a noninvasive but irreversible procedure involving guided, selective irradiation of a targeted substructure, and has been described for the treatment of seizures arising from a temporal focus. Cmelak A. J. et al., Low-dose stereotactic radiosurgery is inadequate for medically intractable mesial temporal lobe epilepsy: a case report, Seizure 10; 442-446; 2001. Heikkinen E. R. et al., Stereotactic radiotherapy instead of conventional epilepsy surgery. A case report, Acta Neurochir (Wien) 119(1-4); 159-160: 1992. The effectiveness of radiotherapy is dose-dependent in both clinical (10 Gy, Heikkinen et al, 1992; 15 Gy, Cmelak et al, 2001) and experimental application (40 Gy, Sun B. et al., Reduction of hippocampal-kindled seizure activity in rats by stereotactic radiosurgery, Exper Neurol 154; 691-695: 1998). Lower dose effects are equivocal while higher doses are effective; however, higher doses also produce histological, irreversible changes not seen at lower doses. Thus, anatomical lesioning of the target structure appears to be important in the efficacy of stereotactic radiosurgery, but may introduce dose-dependent toxicity issues.
Stereotactic procedures can also be used to guide the direct delivery of pharmacologically active agents into specific, localized substructures. These agents may result in either permanent (irreversible) lesions, such as with excitotoxins (e.g. kainic and ibotenic acids), or reversible inactivation, such as with anesthetics and neurotransmitter iontophoresis. Lomber S. G., The advantages and limitations of permanent or reversible deactivation techniques in the assessment of neural function, J Neurosci Meth 86; 109-117: 1999. Delivery may be achieved through both hypodermic cannulae and fine-tipped micropipettes, or variations thereof. Malpeli J. G., Reversible inactivation of subcortical sites by drug injection, J Neurosci Meth 86; 119-128: 1999. Stereotactic placement of electrodes to record intracranial EEG has been described together with simultaneous metabolic assessment through microdialysis sampling of the target. Fried I. et al., Cerebral microdialysis combined with single-neuron and electroencephalographic recording in neurosurgical patients, J Neurosurg 91; 697-705: 1999. Thus, it is possible, to configure a system to assess the neuronal activity within the desired target before, during and after delivery of the pharmacological agent. With any such procedure, assessment of the spatial distribution (spread) of the agent within the target structure can also be determined. Malpeli, 1999, supra; Martin J. H. et al., Pharmacological inactivation in the analysis of the central control of movement, J Neurosci Meth 86; 145-159: 1999. Apart from neuronal activity measured through indwelling depth electrodes, functional (metabolic) assessments can be made with PET imaging to determine the profile and duration of effects of the active agent on the target in question.
In cases where subcortical structures encompass, or are in proximity to, critical functional areas, and thus cannot be approached with standard resective techniques, the treatment regimen may be a maintenance of multiple AEDs for suppression of seizure activity (Kwan and Brodie, 2002 ibid). For functionally critical (eloquent) cortical regions that cannot be approached with standard resective surgical procedures, multiple subpial transections (MST) can be used to treat refractory seizures (Mulligan L. P. et al, Multiple subpial transections: the Yale experience. Epilepsia 42(2):226-229:2001). new approach to the surgical treatment of focal epilepsy, J Neurosurg 70: 231-239:1989) This procedure exploits the natural orthogonal architecture of the cerebral cortex, which is arranged such that functional units are organized vertically while intracortical interconnections are primarily horizontal (perpendicular) to the functional barrels. Synchronous epileptic activity can arise through horizontal intracortical spread. The MST procedure physically disrupts these horizontal interconnections while sparing the vertically-oriented cortical function (Morrell F. et al, Multiple subpial transection: a The MST procedure physically disrupts these horizontal interconnections while sparing the vertically-oriented cortical function (Morrell et al, 1989 ibid). Thus, MST is a surgical technique for intractable epilepsy which reduces the occurrence of electrocorticographical spikes recorded from the cortical surface and can thereby reduce clinical epileptic events. Significantly, MST permits treatment of unresectable epileptogenic foci, such as foci in functionally important (“eloquent”) areas of the cerebral cortex. Resection of eloquent foci can leave an epilepsy patient with a significant reduction in neurological function.
MST has as its' basis the diminution of the spread of electrical activity between nearby superficial cortical neurons and is based upon the anatomic and functional structure of the columnar organization of the higher mammalian neocortex. When used to effectively accomplish a seizure ocus resection, MST reduces the side to side spread of epileptogenic energy along cortical neurons without causing major functional impairment of the centrifugal and centripetal cortical neuronal connections. Thus, MST reduces the horizontal “cross talk” of epileptic discharges in order to prevent the discharge spreading along the cortical service and the resulting seizure.
Brain Motor Systems
Several areas of the cerebrum influence motor activity. Thus, lesion to the motor cortex of the cerebrum, as can result from stoke, can remove inhibition of vestibular and reticular brain stem nuclei, which then become spontaneously active and cause spasm of muscles influenced by, the now disinhibited, lower brain areas.
An accessory motor system of the cerebrum is the basal ganglia. The basal ganglia receives most input from and sends most of its signals back to the cortex. The basal ganglia include the caudate nucleus, putamen, globus pallidus, substantia nigra (which includes the pars compacta) and subthalamic nucleus. Because abnormal signals from the basal ganglia to the motor cortex cause most of the abnormalities in Parkinson's disease, attempts have been made to treat Parkinson's disease by blocking these signals. Thus lesions have been made in the ventrolateral and ventroanterior nuclei of the thalamus to block the feedback circuit from the basal ganglia to the cortex. Additionally, pallidotomy, the surgical ablation of part of the globus pallidus, has been used to effectively treat the motor disorders of Parkinson's disease.
Surgical intervention is believed to assist by interrupting a motoric pathway which, due to a dopaminergic deficiency, had pathologically inhibited the thalamus. The inhibited thalamus in turn understimulates cortical neuronal networks responsible for generating movement. Hence, surgery removes the thalamic inhibition and has been used in the treatment of pharmacoresistant movement disorders. Speelman, J. D., et al., Thalamic Surgery and Tremor, Mov Dis 13(3); 103-106:1998.
Intracranial lesions for the treatment of tremor and other parkinsonian symptoms have been made to the globus pallidus and the ansa lenticularis. Long term results of pallidotomy have sometimes been disappointing. Positive results for the surgical arrest of tremor have been obtained by lesioning the following thalamic nuclei: (1) the ventrointermedius (Vim) or ventral lateral posterior (VLp) nucleus; (2) ventrooralis anterior (Voa) nucleus (Voa and Vop have been collectively termed the ventral lateral anterior nucleus (VLa)); (3) ventrooralis posterior (Vop) nucleus; (4) subthalamic nuclei (campotomy), and; (5) CM-Pf thalamic nuclei. Generally, the ventrolateral thalamus has been the surgical target of choice in the treatment of Parkinson's disease and other systemically administered, drug resistant tremors. Brophy, B. P., et al., Thalamotomy for Parkinsonian Tremor, Stereotact Funct Neurosurg, 69; 1-4:1997. Thalamic excitation of the cortex is necessary for almost all cortical activity.
Stereotactic surgery (assisted by neuroimaging and electrophysiologic recordings) has been used in the management of advanced, pharmacoresistant Parkinson's disease, targeting hyperactive globus pallidus and subthalamic nuclei. An electrode or a probe is placed into the brain using a brain atlas for reference with assistance from brain imaging by computer tomography or magnetic resonance imaging. Lesions in different parts of the pallidum (i.e. posteroventral pallidum), basal ganglia, thalamus and subthalamic nuclei have been carried out to treat motor disorders of Parkinson's disease. Unfortunately, surgical brain lesions create a risk of impairment to speech, visual and cognitive brain areas. Neurotransplantation shows promise but requires further investigation. Additionally, deep brain stimulation using electrodes for the suppression of tremor using can create problems due to wire erosion, lead friction, infection of the implantable pulse generator, malfunction of the implantable pulse generator, electrical shock and lead migration. Other complications due to electrode stimulation can include dysarthria, disequilibrium, paresis and gait disorder. See e.g. Koller, W. C. et al., Surgical Treatment of Parkinson's Disease, J Neurol Sci 167; 1-10:1999, and Schuurman P. R., et al., A Comparison of Continuous Thalamic Stimulation and Thalamotomy for Suppression of Severe Tremor, NEJM 342(7); 461-468:2000.
Aside from surgical ablation or stimulation, external radiotherapy (Gamma Knife Radiosurgery) has also been used to a limited extent for the treatment of drug resistant parkinsonian tremors. Drawbacks with this procedure are that the reduction in tremor is delayed by between one week and eight months after the radiosurgery, and that long term benefits as well as radiation side effects are currently unknown.
As set forth, treatment of parkinsonian tremor and other movement disorders has been carried out by thalamotomy and/or interruption of pallidofugal fibers in the subthalamic region and pallidotomy has also been used. Current concepts of basal ganglia circuitry propose that the loss of striatal dopamine in Parkinson's disease leads to overactivity of the striatal projection to the lateral segment of the globus pallidus. The resulting decrease in lateral pallidal activity results in disinhibition of the subthalamic nucleus, its main projection site. Increased subthalamic activity in turn causes overactivity of the internal segment of the globus pallidus, which projects to the pedunculopontine nucleus (PPN) and the ventrolateral (VL) thalamus. Thus, overactivity in the subthalamic nucleus and internal pallidum produces the parkinsonian symptoms of tremor, bradykinesia and hypokinesia through projections to the PPN and VL thalamus. Lesion in the subthalamic nucleus and the results of pallidotomy, particularly posteroventral pallidotomy, have permitted effective treatment of akinesia in parkinsonian patients.
Botulinum Toxin
The genus Clostridium has more than one hundred and twenty seven species, grouped according to their morphology and functions. The anaerobic, gram positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals referred to as botulism. The spores of Clostridium botulinum are found in soil and can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism. The effects of botulism typically appear 18 to 36 hours after eating the foodstuffs infected with a Clostridium botulinum culture or spores. The botulinum toxin can apparently pass unattenuated through the lining of the gut and attack peripheral motor neurons. Symptoms of botulinum toxin intoxication can progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles and death.
Botulinum toxin type A is the most lethal natural biological agent known to man. About 50 picograms of a commercially available botulinum toxin type A (purified neurotoxin complex)1 is a LD50 in mice (i.e. 1 unit). One unit of BOTOX® (a botulinum toxin type A purified neurotoxin complex, which is also referred to as a botulinum toxin type A complex) contains about 50 picograms (about 56 attomoles) of botulinum toxin type A complex. Interestingly, on a molar basis, botulinum toxin type A is about 1.8 billion times more lethal than diphtheria, about 600 million times more lethal than sodium cyanide, about 30 million times more lethal than cobra toxin and about 12 million times more lethal than cholera. Singh, Critical Aspects of Bacterial Protein Toxins, pages 63-84 (chapte r 4) of Natural Toxins II, edited by B. R. Singh et al., Plenum Press, New York (1976) (where the stated LD50 of botulinum toxin type A of 0.3 ng equals 1 U is corrected for the fact that about 0.05 ng of BOTOX® (a botulinum toxin type A complex) equals 1 unit). One unit (U) of botulinum toxin is defined as the LD50 upon intraperitoneal injection into female Swiss Webster mice weighing 18 to 20 grams each. 1Available from Allergan, Inc., of Irvine, Calif. under the tradename BOTOX® (a botulinum toxin type A complex) in 100 unit vials.
Seven immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum neurotoxin serotypes A, B, C1, D, E, F and G each of which is distinguished by neutralization with type-specific antibodies. The different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. For example, it has been determined that botulinum toxin type A is 500 times more potent, as measured by the rate of paralysis produced in the rat, than is botulinum toxin type B. Additionally, botulinum toxin type B has been determined to be non-toxic in primates at a dose of 480 U/kg which is about 12 times the primate LD50 for botulinum toxin type A. Moyer E et al., Botulinum Toxin Type B: Experimental and Clinical Experience, being chapter 6, pages 71-85 of “Therapy With Botulinum Toxin”, edited by Jankovic, J. et al. (1994), Marcel Dekker, Inc. Botulinum toxin apparently binds with high affinity to cholinergic motor neurons, is translocated into the neuron and blocks the release of acetylcholine.
Regardless of serotype, the molecular mechanism of toxin intoxication appears to be similar and to involve at least three steps or stages. In the first step of the process, the toxin binds to the presynaptic membrane of the target neuron through a specific interaction between the heavy chain, H chain, and a cell surface receptor; the receptor is thought to be different for each type of botulinum toxin and for tetanus toxin. The carboxyl end segment of the H chain, HC, appears to be important for targeting of the toxin to the cell surface.
In the second step, the toxin crosses the plasma membrane of the poisoned cell. The toxin is first engulfed by the cell through receptor-mediated endocytosis, and an endosome containing the toxin is formed. The toxin then escapes the endosome into the cytoplasm of the cell. This step is thought to be mediated by the amino end segment of the H chain, HN, which triggers a conformational change of the toxin in response to a pH of about 5.5 or lower. Endosomes are known to possess a proton pump which decreases intra-endosomal pH. The conformational shift exposes hydrophobic residues in the toxin, which permits the toxin to embed itself in the endosomal membrane. The toxin (or at a minimum the light chain) then translocates through the endosomal membrane into the cytoplasm.
The last step of the mechanism of botulinum toxin activity appears to involve reduction of the disulfide bond joining the heavy chain, H chain, and the light chain, L chain. The entire toxic activity of botulinum and tetanus toxins is contained in the L chain of the holotoxin; the L chain is a zinc (Zn++) endopeptidase which selectively cleaves proteins essential for recognition and docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the vesicles with the plasma membrane. Tetanus neurotoxin, botulinum toxin types B, D, F, and G cause degradation of synaptobrevin (also called vesicle-associated membrane protein (VAMP)), a synaptosomal membrane protein. Most of the VAMP present at the cytoplasmic surface of the synaptic vesicle is removed as a result of any one of these cleavage events. Botulinum toxin serotype A and E cleave SNAP-25. Botulinum toxin serotype C1 was originally thought to cleave syntaxin, but was found to cleave syntaxin and SNAP-25. Each of the botulinum toxins specifically cleaves a different bond, except botulinum toxin type B (and tetanus toxin) which cleave the same bond.
Although all the botulinum toxins serotypes apparently inhibit release of the neurotransmitter acetylcholine at the neuromuscular junction, they do so by affecting different neurosecretory proteins and/or cleaving these proteins at different sites. For example, botulinum types A and E both cleave the 25 kiloDalton (kD) synaptosomal associated protein (SNAP-25), but they target different amino acid sequences within this protein. Botulinum toxin types B, D, F and G act on vesicle-associated protein (VAMP, also called synaptobrevin), with each serotype cleaving the protein at a different site. Finally, botulinum toxin type C1 has been shown to cleave both syntaxin and SNAP-25. These differences in mechanism of action may affect the relative potency and/or duration of action of the various botulinum toxin serotypes. Apparently, a substrate for a botulinum toxin can be found in a variety of different cell types. See e.g. Gonelle-Gispert, C., et al., SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion, Biochem J. 1; 339 (pt 1):159-65:1999, and Boyd R. S. et al., The effect of botulinum neurotoxin-B on insulin release from a ∃-cell line, and Boyd R. S. et al., The insulin secreting ∃-cell line, HIT-15, contains SNAP-25 which is a target for botulinum neurotoxin-A, both published at Mov Disord, 10(3):376:1995 (pancreatic islet B cells contains at least SNAP-25 and synaptobrevin).
The molecular weight of the botulinum toxin protein molecule, for all seven of the known botulinum toxin serotypes, is about 150 kD. Interestingly, the botulinum toxins are released by Clostridial bacterium as complexes comprising the 150 kD botulinum toxin protein molecule along with associated non-toxin proteins. Thus, the botulinum toxin type A complex can be produced by Clostridial bacterium as 900 kD, 500 kD and 300 kD forms. Botulinum toxin types B and C1 is apparently produced as only a 700 kD or 500 kD complex. Botulinum toxin type D is produced as both 300 kD and 500 kD complexes. Finally, botulinum toxin types E and F are produced as only approximately 300 kD complexes. The complexes (i.e. molecular weight greater than about 150 kD) are believed to contain a non-toxin hemaglutinin protein and a non-toxin and non-toxic nonhemaglutinin protein. These two non-toxin proteins (which along with the botulinum toxin molecule comprise the relevant neurotoxin complex) may act to provide stability against denaturation to the botulinum toxin molecule and protection against digestive acids when toxin is ingested. Additionally, it is possible that the larger (greater than about 150 kD molecular weight) botulinum toxin complexes may result in a slower rate of diffusion of the botulinum toxin away from a site of intramuscular injection of a botulinum toxin complex.
All the botulinum toxin serotypes are made by Clostridium botulinum bacteria as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that make botulinum toxin serotypes A and G possess endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures in predominantly their active form. In contrast, botulinum toxin serotypes C1, D, and E are synthesized by nonproteolytic strains and are therefore typically unactivated when recovered from culture. Serotypes B and F are produced by both proteolytic and nonproteolytic strains and therefore can be recovered in either the active or inactive form. However, even the proteolytic strains that produce, for example, the botulinum toxin type B serotype only cleave a portion of the toxin produced. The exact proportion of nicked to unnicked molecules depends on the length of incubation and the temperature of the culture. Therefore, a certain percentage of any preparation of, for example, the botulinum toxin type B toxin is likely to be inactive, possibly accounting for a lower potency of botulinum toxin type B as compared to botulinum toxin type A. The presence of inactive botulinum toxin molecules in a clinical preparation will contribute to the overall protein load of the preparation, which has been linked to increased antigenicity, without contributing to its clinical efficacy.
Botulinum toxins and toxin complexes can be obtained from, for example, List Biological Laboratories, Inc., Campbell, Calif.; the Centre for Applied Microbiology and Research, Porton Down, U.K.; Wako (Osaka, Japan), as well as from Sigma Chemicals of St Louis, Mo. Commercially available botulinum toxin containing pharmaceutical compositions include BOTOX® (Botulinum toxin type A neurotoxin complex with human serum albumin and sodium chloride) available from Allergan, Inc., of Irvine, Calif. in 100 unit vials as a lyophilized powder to be reconstituted with 0.9% sodium chloride before use), Dysport® (Clostridium botulinum type A toxin haemagglutinin complex with human serum albumin and lactose in the formulation), available from Ipsen Limited, Berkshire, U.K. as a powder to be reconstituted with 0.9% sodium chloride before use), and MyoBloc™ (an injectable solution comprising botulinum toxin type B, human serum albumin, sodium succinate, and sodium chloride at about pH 5.6, available from Elan Corporation, Dublin, Ireland).
The success of botulinum toxin type A to treat a variety of clinical conditions has led to interest in other botulinum toxin serotypes. Additionally, pure botulinum toxin has been used to treat humans. see e.g. Kohl A., et al., Comparison of the effect of botulinum toxin A (Botox (R)) with the highly-purified neurotoxin (NT 201) in the extensor digitorum brevis muscle test, Mov Disord 2000; 15(Suppl 3):165. Hence, a pharmaceutical composition can be prepared using a pure botulinum toxin.
The type A botulinum toxin is known to be soluble in dilute aqueous solutions at pH 4-6.8. At pH above about 7 the stabilizing nontoxic proteins dissociate from the neurotoxin, resulting in a gradual loss of toxicity, particularly as the pH and temperature rise. Schantz E. J., et al Preparation and characterization of botulinum toxin type A for human treatment (in particular pages 44-45), being chapter 3 of Jankovic, J., et al, Therapy with Botulinum Toxin, Marcel Dekker, Inc (1994).
The botulinum toxin molecule (about 150 kDa), as well as the botulinum toxin complexes (about 300-900 kDa), such as the toxin type A complex are also extremely susceptible to denaturation due to surface denaturation, heat, and alkaline conditions. Inactivated toxin forms toxoid proteins which may be immunogenic. The resulting antibodies can render a patient refractory to toxin injection.
In vitro studies have indicated that botulinum toxin inhibits potassium cation induced release of both acetylcholine and norepinephrine from primary cell cultures of brainstem tissue. Additionally, it has been reported that botulinum toxin inhibits the evoked release of both glycine and glutamate in primary cultures of spinal cord neurons and that in brain synaptosome preparations botulinum toxin inhibits the release of each of the neurotransmitters acetylcholine, dopamine, norepinephrine (Habermann E., et al., Tetanus Toxin and Botulinum A and C Neurotoxins Inhibit Noradrenaline Release From Cultured Mouse Brain, J Neurochem 51(2); 522-527:1988) CGRP, substance P and glutamate (Sanchez-Prieto, J., et al., Botulinum Toxin A Blocks Glutamate Exocytosis From Guinea Pig Cerebral Cortical Synaptosomes, Eur J. Biochem 165; 675-681:1987. Thus, when adequate concentrations are used, stimulus-evoked release of most neurotransmitters is blocked by botulinum toxin. See e.g. Pearce, L. B., Pharmacologic Characterization of Botulinum Toxin For Basic Science and Medicine, Toxicon 35(9); 1373-1412 at 1393; Bigalke H., et al., Botulinum A Neurotoxin Inhibits Non-Cholinergic Synaptic Transmission in Mouse Spinal Cord Neurons in Culture, Brain Research 360; 318-324:1985; Habermann E., Inhibition by Tetanus and Botulinum A Toxin of the release of [3H]Noradrenaline and [3H]GABA From Rat Brain Homogenate, Experientia 44; 224-226:1988, Bigalke H., et al., Tetanus Toxin and Botulinum A Toxin Inhibit Release and Uptake of Various Transmitters, as Studied with Particulate Preparations From Rat Brain and Spinal Cord, Naunyn-Schmiedeberg's Arch Pharmacol 316; 244-251:1981, and; Jankovic J. et al., Therapy With Botulinum Toxin, Marcel Dekker, Inc., (1994), page 5.
Botulinum toxin type A can be obtained by establishing and growing cultures of Clostridium botulinum in a fermenter and then harvesting and purifying the fermented mixture in accordance with known procedures. All the botulinum toxin serotypes are initially synthesized as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that make botulinum toxin serotypes A and G possess endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures in predominantly their active form. In contrast, botulinum toxin serotypes C1, D and E are synthesized by nonproteolytic strains and are therefore typically unactivated when recovered from culture. Serotypes B and F are produced by both proteolytic and nonproteolytic strains and therefore can be recovered in either the active or inactive form. However, even the proteolytic strains that produce, for example, the botulinum toxin type B serotype only cleave a portion of the toxin produced. The exact proportion of nicked to unnicked molecules depends on the length of incubation and the temperature of the culture. Therefore, a certain percentage of any preparation of, for example, the botulinum toxin type B toxin is likely to be inactive, possibly accounting for the known significantly lower potency of botulinum toxin type B as compared to botulinum toxin type A. The presence of inactive botulinum toxin molecules in a clinical preparation will contribute to the overall protein load of the preparation, which has been linked to increased antigenicity, without contributing to its clinical efficacy. Additionally, it is known that botulinum toxin type B has, upon intramuscular injection, a shorter duration of activity and is also less potent than botulinum toxin type A at the same dose level.
High quality crystalline botulinum toxin type A can be produced from the Hall A strain of Clostridium botulinum with characteristics of ≧3×107 U/mg, an A260/A278 of less than 0.60 and a distinct pattern of banding on gel electrophoresis. The known Schantz process can be used to obtain crystalline botulinum toxin type A, as set forth in Schantz, E. J., et al, Properties and use of Botulinum toxin and Other Microbial Neurotoxins in Medicine, Microbiol Rev. 56; 80-99:1992. Generally, the botulinum toxin type A complex can be isolated and purified from an anaerobic fermentation by cultivating Clostridium botulinum type A in a suitable medium. The known process can also be used, upon separation out of the non-toxin proteins, to obtain pure botulinum toxins, such as for example: purified botulinum toxin type A with an approximately 150 kD molecular weight with a specific potency of 1-2×108 LD50 U/mg or greater; purified botulinum toxin type B with an approximately 156 kD molecular weight with a specific potency of 1-2×108 LD50 U/mg or greater, and; purified botulinum toxin type F with an approximately 155 kD molecular weight with a specific potency of 1-2×107 LD50 U/mg or greater.
Either the pure botulinum toxin (i.e. the 150 kilodalton botulinum toxin molecule) or the toxin complex can be used to prepare a pharmaceutical composition. Both molecule and complex are susceptible to denaturation due to surface denaturation, heat, and alkaline conditions. Inactivated toxin forms toxoid proteins which may be immunogenic. The resulting antibodies can render a patient refractory to toxin injection.
As with enzymes generally, the biological activities of the botulinum toxins (which are intracellular peptidases) is dependant, at least in part, upon their three dimensional conformation. Thus, botulinum toxin type A is detoxified by heat, various chemicals surface stretching and surface drying. Additionally, it is known that dilution of the toxin complex obtained by the known culturing, fermentation and purification to the much, much lower toxin concentrations used for pharmaceutical composition formulation results in rapid detoxification of the toxin unless a suitable stabilizing agent is present. Dilution of the toxin from milligram quantities to a solution containing nanograms per milliliter presents significant difficulties because of the rapid loss of specific toxicity upon such great dilution. Since the toxin may be used months or years after the toxin containing pharmaceutical composition is formulated, the toxin can stabilized with a stabilizing agent such as albumin and gelatin.
A commercially available botulinum toxin containing pharmaceutical composition is sold under the trademark BOTOX® (available from Allergan, Inc., of Irvine, Calif.). BOTOX® (a botulinum toxin type A complex) consists of a purified botulinum toxin type A complex, albumin and sodium chloride packaged in sterile, vacuum-dried form. The botulinum toxin type A is made from a culture of the Hall strain of Clostridium botulinum grown in a medium containing N-Z amine and yeast extract. The botulinum toxin type A complex is purified from the culture solution by a series of acid precipitations to a crystalline complex consisting of the active high molecular weight toxin protein and an associated hemagglutinin protein. The crystalline complex is re-dissolved in a solution containing saline and albumin and sterile filtered (0.2 microns) prior to vacuum-drying. The vacuum-dried product is stored in a freezer at or below−5° C. BOTOX® (a botulinum toxin type A complex) can be reconstitued with sterile, non-preserved saline prior to intramuscular injection. Each vial of BOTOX® (a botulinum toxin type A complex) contains about 100 units (U) of Clostridium botulinum toxin type A purified neurotoxin complex, 0.5 milligrams of human serum albumin and 0.9 milligrams of sodium chloride in a sterile, vacuum-dried form without a preservative.
To reconstitute vacuum-dried BOTOX® (a botulinum toxin type A complex), sterile normal saline without a preservative; (0.9% Sodium Chloride Injection) is used by drawing up the proper amount of diluent in the appropriate size syringe. Since BOTOX® (a botulinum toxin type A complex) may be dispersed or denatured by bubbling or similar violent agitation, the diluent is gently injected into the vial. For sterility reasons BOTOX® (a botulinum toxin type A complex) is preferably administered within four hours after the vial is removed from the freezer and reconstituted. During these four hours, reconstituted BOTOX® (a botulinum toxin type A complex) can be stored in a refrigerator at about 2° C. to about 8° C. Reconstituted, refrigerated BOTOX® (a botulinum toxin type A complex) has been reported to retain its potency for at least about two weeks. Neurology, 48:249-53:1997.
Botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles. Botulinum toxin type A (BOTOX®, a botulinum toxin type A complex) was approved by the U.S. Food and Drug Administration in 1989 for the treatment of essential blepharospasm, strabismus and hemifacial spasm in patients over the age of twelve. In 2000 the FDA approved commercial preparations of type A (BOTOX®, a botulinum toxin type A complex) and type B botulinum toxin MYOBLOC® (a botulinum toxin type B preparation) serotypes for the treatment of cervical dystonia, and in 2002 the FDA approved a type A botulinum toxin (BOTOX®, a botulinum toxin type A complex) for the cosmetic treatment of certain hyperkinetic (glabellar) facial wrinkles. Clinical effects of peripheral intramuscular botulinum toxin type A are usually seen within one week of injection and sometimes within a few hours. The typical duration of symptomatic relief (i.e. flaccid muscle paralysis) from a single intramuscular injection of botulinum toxin type A can be about three months, although in some cases the effects of a botulinum toxin induced denervation of a gland, such as a salivary gland, have been reported to last for several years. For example, it is known that botulinum toxin type A can have an efficacy for up to 12 months (Naumann M, et al., Botulinum toxin type A in the treatment of focal, axillary and palmar hyperhidrosis and other hyperhidrotic conditions, European J. Neurology 6 (Supp 4): S111-S115:1999), and in some circumstances for as long as 27 months. Ragona, R. M, et al., Management of parotid sialocele with botulinum toxin, The Laryngoscope 109:1344-1346:1999. However, the usual duration of an intramuscular injection of Botox® is typically about 3 to 4 months.
It has been reported that botulinum toxin type A has been used in clinical settings as follows:    (1) about 75-125 units of BOTOX® (a botulinum toxin type A complex) per intramuscular injection (multiple muscles) to treat cervical dystonia;    (2) 5-10 units of BOTOX® (a botulinum toxin type A complex) per intramuscular injection to treat glabellar lines (brow furrows) (5 units injected intramuscularly into the procerus muscle and 10 units injected intramuscularly into each corrugator supercilii muscle);    (3) about 30-80 units of BOTOX® (a botulinum toxin type A complex) to treat constipation by intrasphincter injection of the puboreetalis muscle;    (4) about 1-5 units per muscle of intramuscularly injected BOTOX® (a botulinum toxin type A complex) to treat blepharospasm by injecting the lateral pre-tarsal orbicularis oculi muscle of the upper Lid and the lateral pre-tarsal orbicularis oculi of the lower lid.    (5) to treat strabismus, extraocular muscles have been injected intramuscularly with between about 1-5 units of BOTOX® (a botulinum toxin type A complex) the amount injected varying based upon both the size of the muscle to be injected and the extent of muscle paralysis desired (i.e. amount of diopter correction desired).    (6) to treat upper limb spasticity following stroke by intramuscular injections of BOTOX® (a botulinum toxin type A complex) into five different upper limb flexor muscles, as follows:    (a) flexor digitorum profundus: 7.5 U to 30 U    (b) flexor digitorum sublimus: 7.5 U to 30 U    (c) flexor carpi ulnaris: 10 U to 40 U    (d) flexor carpi radialis: 15 U to 60 U    (e) biceps brachui: 50 U to 200 U. Each of the five indicated muscles has been injected at the same treatment session, so that the patient receives from 90 U to 360 U of upper limb flexor muscle BOTOX® (a botulinum toxin type A complex) by intramuscular injection at each treatment session.    (7) to treat migraine, pericranial injected (injected symmetrically into glabellar, frontalis and temporalis muscles) injection of 25 U of BOTOX® (a botulinum toxin type A complex) has showed significant benefit as a prophylactic treatment of migraine compared to vehicle as measured by decreased measures of migraine frequency, maximal severity, associated vomiting and acute medication use over the three month period following the 25 U injection.
Additionally, intramuscular botulinum toxin has been used in the treatment of tremor in patients with Parkinson's disease, although it has been reported that results have not been impressive. Marjama-Lyons, J., et al., Tremor-Predominant Parkinson's Disease, Drugs & Aging 16(4); 273-278:2000.
Treatment of certain gastrointestinal and smooth muscle disorders with a botulinum toxin are known. See e.g. U.S. Pat. Nos. 5,427,291 and 5,674,205 (Pasricha). Additionally, transurethral injection of a botulinum toxin into a bladder sphincter to treat a urination disorder is known (see e.g. Dykstra, D. D., et al, Treatment of detrusor-sphincter dyssynergia with botulinum A toxin: A double-blind study, Arch Phys Med Rehabil 1990 January; 71:24-6), as is injection of a botulinum toxin into the prostate to treat prostatic hyperplasia. See e.g. U.S. Pat. No. 6,365,164 (Schmidt).
U.S. Pat. No. 5,766,605 (Sanders) proposes the treatment of various autonomic disorders, such as hypersalivation and rhinittis, with a botulinum toxin.
Furthermore, various afflictions, such as hyperhydrosis and headache, treatable with a botulinum toxin are discussed in WO 95/17904 (PCT/US94/14717) (Aoki). EP 0 605 501 B1 (Graham) discusses treatment of cerebral palsy with a botulinum toxin and U.S. Pat. No. 6,063,768 (First) discusses treatment of neurogenic inflammation with a botulinum toxin.
Botulinum toxin has been used to study the release of dopamine from various brain cells, in light of the theory that Parkinson's disease is due to the death of dopamine releasing cell in the striatal region of the brain. See e.g. Bergquist F. et al., Evidence for different exocytosis pathways in dendritic and terminal dopamine release in vivo, Brain Research 950; 245-253: (2002), which me determined that in vivo, intracranial microdialysis of a botulinum toxin type A into either the substantia nigra or striatum use of rats strongly reduced release of somatodentritic dopamine from cells therein. Hence, one could conclude that evidence that the death of dopamine releasing cells in either the substantia nigra or substantia striatum regions of the brain can be a factor in the occurrence of Parkinson's disease.
In addition to having pharmacologic actions at the peripheral location, botulinum toxins can also have inhibitory effects in the central nervous system. Work by Weigand et al, (125I-labelled botulinum A neurotoxin:pharmacokinetics in cats after intramuscular injection, Nauny-Schmiedeberg's Arch. Pharmacol. 1976; 292,161-165), and Habermann, (125I-labelled Neurotoxin from clostridium botulinum A: preparation, binding to synaptosomes and ascent to the spinal cord, Nauny-Schmiedeberg's Arch. Pharmacol. 1974; 281, 47-56) showed that botulinum toxin is able to ascend to the spinal area by retrograde transport. As such, a botulinum toxin injected at a peripheral location, for example intramuscularly, may be retrograde transported to the spinal cord.
In vitro studies have indicated that botulinum toxin inhibits potassium cation induced release of both acetylcholine and norepinephrine from primary cell cultures of brainstem tissue. Additionally, it has been reported that botulinum toxin inhibits the evoked release of both glycine and glutamate in primary cultures of spinal cord neurons and that in brain synaptosome preparations botulinum toxin inhibits the release of each of the neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP and glutamate.
U.S. Pat. No. 5,989,545 discloses that a modified clostridial neurotoxin or fragment thereof, preferably a botulinum toxin, chemically conjugated or recombinantly fused to a particular targeting moiety can be used to treat pain by administration of the agent to the spinal cord.
Tetanus toxin, as wells as derivatives (i.e. with a non-native targeting moiety), fragments, hybrids and chimeras thereof can also have therapeutic utility. The tetanus toxin bears many similarities to the botulinum toxins. Thus, both the tetanus toxin and the botulinum toxins are polypeptides made by closely related species of Clostridium (Clostridium tetani and Clostridium botulinum, respectively). Additionally, both the tetanus toxin and the botulinum toxins are dichain proteins composed of a light chain (molecular weight about 50 kD) covalently bound by a single disulfide bond to a heavy chain (molecular weight about 100 kD). Hence, the molecular weight of tetanus toxin and of each of the seven botulinum toxins (non-complexed) is about 150 kD. Furthermore, for both the tetanus toxin and the botulinum toxins, the light chain bears the domain which exhibits intracellular biological (protease) activity, while the heavy chain comprises the receptor binding (immunogenic) and cell membrane translocational domains.
Further, both the tetanus toxin and the botulinum toxins exhibit a high, specific affinity for gangliocide receptors on the surface of presynaptic cholinergic neurons. Receptor mediated endocytosis of tetanus toxin by peripheral cholinergic neurons results in retrograde axonal transport, blocking of the release of inhibitory neurotransmitters from central synapses and a spastic paralysis. Contrarily, receptor mediated endocytosis of botulinum toxin by peripheral cholinergic neurons results in little if any retrograde transport, inhibition of acetylcholine exocytosis from the intoxicated peripheral motor neurons and a flaccid paralysis.
The tetanus toxin and the botulinum toxins resemble each other in both biosynthesis and molecular architecture. Thus, there is an overall 34% identity between the protein sequences of tetanus toxin and botulinum toxin type A, and a sequence identity as high as 62% for some functional domains. Binz T. et al., The Complete Sequence of Botulinum Neurotoxin Type A and Comparison with Other Clostridial Neurotoxins, J Biological Chemistry 265(16); 9153-9158:1990.
It is known that injection of tetanus toxin into rat cerebral cortex can induce chronic epilepsy and based upon this finding tetanus toxin has been used to provide a model of focal epilepsies. See e.g. Mellanby, J., Tetanus toxin as a tool for investigating the consequences of excessive neuronal excitation, pages 291-297 of Dasgupta, B. R., Botulinum and tetanus neurotoxins, Plenum Press (1993), and; Jefferys, J. G., et al., Chronic focal epilepsy induced by intracerebral tetanus toxin, Ital J Neurol Sci 1995 February-March; 16(1-2):27-32.
Contrary to the result upon intracranial administration of a tetanus toxin, seizures do not result upon intrathecal administration of a botulinum toxin to a mammal (see e.g. U.S. Pat. No. 6,113,915). Additionally, and as set forth above, intoxication with tetanus toxin results in a spastic paralysis, whereas intoxication with a botulinum toxin results in a flaccid paralysis. Intramuscular administration of a botulinum toxin to treat epilepsy has been reported to be ineffective. Tarsy, D. et al., Botulinum toxin treatment is not effective for epilepsy partialis continua, Mov Disord 1995; 10(3):357-8.
Additionally, intraventricular injection of tetanus toxin can cause an increase in serotonin levels in rats. Aguilera, J. et al., Stereotaxic injection of tetanus toxin in rat central nervous system causes alteration in normal levels of monoamines, J. Neurochem, 56(3); 733-738: 1991.
Acetylcholine
Typically only a single type of small molecule neurotransmitter is released by each type of neuron in the mammalian nervous system. The neurotransmitter acetylcholine is secreted by neurons in many areas of the brain, but specifically by the large pyramidal cells of the motor cortex, by several different neurons in the basal ganglia, by the motor neurons that innervate the skeletal muscles, by the preganglionic neurons of the autonomic nervous system (both sympathetic and parasympathetic), by the postganglionic neurons of the parasympathetic nervous system, and by some of the postganglionic neurons of the sympathetic nervous system. Essentially, only the postganglionic sympathetic nerve fibers to the sweat glands, the piloerector muscles and a few blood vessels are cholinergic, as most of the postganglionic neurons of the sympathetic nervous system secret the neurotransmitter norepinephrine. In most instances acetylcholine has an excitatory effect. However, acetylcholine is known to have inhibitory effects at some of the peripheral parasympathetic nerve endings, such as inhibition of heart rate by the vagal nerve.
The efferent signals of the autonomic nervous system are transmitted to the body through either the sympathetic nervous system or the parasympathetic nervous system. The preganglionic neurons of the sympathetic nervous system extend from preganglionic sympathetic neuron cell bodies located in the intermediolateral horn of the spinal cord. The preganglionic sympathetic nerve fibers, extending from the cell body, synapse with postganglionic neurons located in either a paravertebral sympathetic ganglion or in a prevertebral ganglion. Since, the preganglionic neurons of both the sympathetic and parasympathetic nervous system are cholinergic, application of acetylcholine to the ganglia will excite both sympathetic and parasympathetic postganglionic neurons.
Acetylcholine activates two types of receptors, muscarinic and nicotinic receptors. The muscarinic receptors are found in all effector cells stimulated by the postganglionic, neurons of the parasympathetic nervous system as well as in those stimulated by the postganglionic cholinergic neurons of the sympathetic nervous system. The nicotinic receptors are found in the adrenal medulla, as well as within the autonomic ganglia, that is on the cell surface of the postganglionic neuron at the synapse between the preganglionic and postganglionic neurons of both the sympathetic and parasympathetic systems. Nicotinic receptors are also found in many nonautonomic nerve endings, for example in the membranes of skeletal muscle fibers at the neuromuscular junction.
Acetylcholine is released from cholinergic neurons when small, clear, intracellular vesicles fuse with the presynaptic neuronal cell membrane. A wide variety of non-neuronal secretory cells, such as, adrenal medulla (as well as the PC12 cell line) and pancreatic islet cells release catecholamines and parathyroid hormone, respectively, from large dense-core vesicles. The PC12 cell line is a clone of rat pheochromocytoma cells extensively used as a tissue culture model for studies of sympathoadrenal development. Botulinum toxin inhibits the release of both types of compounds from both types of cells in vitro, permeabilized (as by electroporation) or by direct injection of the toxin into the denervated cell. Botulinum toxin is also known to block release of the neurotransmitter glutamate from cortical synaptosomes cell cultures.
A neuromuscular junction is formed in skeletal muscle by the proximity of axons to muscle cells. A signal transmitted through the nervous system results in an action potential at the terminal axon, with activation of ion channels and resulting release of the neurotransmitter acetylcholine from intraneuronal synaptic vesicles, for example at the motor endplate of the neuromuscular junction. The acetylcholine crosses the extracellular space to bind with acetylcholine receptor proteins on the surface of the muscle end plate. Once sufficient binding has occurred, an action potential of the muscle cell causes specific membrane ion channel changes, resulting in muscle cell contraction. The acetylcholine is then released from the muscle cells and metabolized by cholinesterases in the extracellular space. The metabolites are recycled back into the terminal axon for reprocessing into further acetylcholine.
Cholinergic Brain Systems
Cholinergic influence of both the motor and visual thalamus originates from both the brainstem and the basal forebrain. See e.g. Billet S., et al., Cholinergic Projections to the Visual Thalamus and Superior Colliculus, Brain Res. 847; 121-123:1999 and Oakman, S. A. et al., Characterization of the Extent of Pontomesencephalic Cholinergic Neurons' projections to the Thalamus: Comparison with Projections to Midbrain Dopaminergic Groups, Neurosci 94(2); 529-547; 1999. Thus, it is known based on histochemical studies using acetylcholinesterase (AchE) staining and retrograde tracing with choline acetyltransferase (ChAT) immunochemistry that there can be ascending cholinergic stimulation by the brainstem of thalamic neurons. Steriade M. et al., Brain Cholinergic Systems, Oxford University Press (1990), chapter 1. Indeed, many thalamic nuclei receive dense cholinergic innervation from brainstem reticular formations. Ibid, page 167. Known brainstem cholinergic cell groups are located within: (1) the rostral pons at what is termed a Ch5 location, which is located within the central tegmental field around the brachium conjunctivum, forming a pedunculopontine tegmental nucleus, and; (2) the caudal part of the midbrain, at what is termed a Ch6 location, the laterodorsal tegmental nucleus, which is embedded in the periaqueductal and periventricular gray matter. The Ch5 and Ch6 cell groups can consist almost exclusively of cholinergic neurons and together form the pontine cholinergic system. The Ch5-Ch6 cholinergic groups provide direct ascending projections that terminate in a number of target structure in the midbrain, diencephalon and telencephalon, including the superior colliculus, anterior pretectal area, interstitial magnocellular nucleus of the posterior commissure, lateral habenular nucleus, thalamus, magnocellular preoptic nucleus, lateral mammillary nucleus, basal forebrain, olfactory bulb, medial prefrontal cortex and pontine nuclei. Stone T. W., CNS Neurotransmitters and Neuromodulators: Acetylcholine, CRC Press (1995), page 16. See also Schafer M. K.-H. et al., Cholinergic Neurons and Terminal Fields Revealed by Immunochemistry for the Vesicular Acetylcholine Transporter. I. Central Nervous System, Neuroscience, 84(2); 331-359:1998. Three dimensional localization of Ch1-8 cholinergic nuclei have been mapped in humans. See e.g. Tracey, D. J., et al., Neurotransmitters in the Human Brain, Plenum Press (1995), pages 136-139.
Additionally, the basal forebrain (proencephalon) provides cholinergic innervation of the dorsal thalamus, as well as to the neocortex, hippocampus, amygdala and olfactory bulb. See e.g. Steridae, page 136-136, supra. Basal forebrain areas where the great proportion of neurons are cholinergic include the medial septal nucleus (Ch1), the vertical branches of the diagonal band nuclei (Ch2), the horizontal branches of the diagonal band nuclei (Ch3), and the magnocellular nucleus basalis (Ch4), which is located dorsolaterally to the Ch3 cell group. Ch1 and Ch2 provide the major component of cholinergic projection to the hippocampus. The cells in the Ch3 sector project to the olfactory bulb.
Furthermore, cholinergic neurons are present in the thalamus. Rico, B. et al., A Population of Cholinergic Neurons is Present in the Macaque Monkey Thalamus, Eur J Neurosci, 10; 2346-2352:1998.
Abnormalities in the brain's cholinergic system have been consistently identified in a variety of neuropsychiatric disorders including Alzheimer's disease, Parkinson's disease and dementia with Lewy bodies. Thus, in Alzheimer's disease there is hypoactivity of cholinergic projections to the hippocampus and cortex. In individuals with dementia with Lewy bodies extensive neocortical cholinergic deficits are believed to exist and in Parkinson's disease there is a loss of pedunculopontine cholinergic neurons. Notably, in vivo imaging of cholinergic activity in the human brain has been reported. Perry, et al., Acetylcholine in Mind: a Neurotransmitter Correlate of Consciousness?, TINS 22(6); 273-280:1999
As set forth, current therapies relating to neuronal inhibition, such as resection radiosurgery ablation, microinjection of pharmacological agents confirm that suppression of an epileptogenic focus can arrest seizures. Unfortunately such methods of treating focal epilepsy either provide transient effects, irreversible damage to brain tissue or introduce unwanted toxicity.
What is needed therefore is an intracranial method for effectively treating focal epilepsies by administration of a pharmaceutical which has the characteristics of long duration of activity, low rates of diffusion out of a chosen intracranial target tissue where administered, and nominal systemic effects at therapeutic dose levels.